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  • Electronic Resource  (2)
  • Bent DNA  (1)
  • Chloroplast 16s ribosomal RNA  (1)
  • 1
    ISSN: 1432-0983
    Keywords: Nicotiana plumbaginifolia ; Chloroplast 16s ribosomal RNA ; Streptomycin resistance
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In a previous paper we reported the isolation of streptomycin-resistant mutants from Nicotiana plumbaginifolia and presented evidence for chloroplast control of the resistance trait. To understand the molecular basis of the resistance in these mutants, we sequenced three regions in the chloroplast 16s rRNA gene, which correspond to the 5′ terminus, the 530 loop, and the 900 stem/loop of Escherichia coli 16s rRNA, and compared them with the sequences of the wild-type. Our results show that: (1) nine mutants have a C to T change at position 912, (2) one mutant (SR1021) has a G to A change at position 885, (3) one mutant has a C to T change at position 526, based on E. coli numbering; and (4) three mutants do not have any change in the regions analyzed. The point mutation detected in SR1021 has not been reported previously. In E. coli 16s rRNA, position 885 is protected from chemical probing by ribosomal protein S12 and is closely juxtaposed with the streptomycin-binding region (positions 912–915) in the predicted secondary structure. It is likely that the G to A transition at this position is a novel mutation for streptomycin resistance.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1617-4623
    Keywords: Chloroplast ; Replication origin ; Bent DNA
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary We demonstrated that the 1055 by restriction fragment containing OriA, a chloroplast DNA replication origin of Chlamydomonas reinhardtii, has electrophoretic anomalies characteristic of bent DNA. A tandem dimer of the region was constructed. Quantitative measurement of the relative gel mobility of a set of permuted fragments was used to extrapolate the approximate position of the bent DNA segment. By analyzing the gel mobility of short, sequenced fragments of the bent DNA region, the putative bending locus was identified. Two A4 tracts and two A5 tracts were located in the bending locus. Oligonucleotide-directed mutagenesis was then used to disrupt the A tract or the spacing between A tracts and the effect of site-specific mutation on electrophoretic mobility was analyzed. To assess the functional role of the bent DNA region, subclones containing the bending locus, mutated bending locus, and regions flanking the bending locus were constructed. Each subclone was used as template in an in vitro DNA replication system which preferentially initiated DNA replication at OriA. A 224 by subclone with the bending locus positioned in the middle displayed the highest replication function and was sufficient to initiate DNA replication in vitro. Site-specific mutations or alterations of the A tracts resulted in decreased DNA bending and decreased DNA replication activity.
    Type of Medium: Electronic Resource
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